The In Vitro Core will provide the centralized bacterial, cell culture and molecular microbiology diagnostic support needed for each of the Projects. Cp and Pg will be grown in high-volume at high titter for in-vivo inoculation experiments, and for in-vitro cell stimulation experiments. Purified Cp will be prepared in the appropriate cell lines to avoid cross species antigen stimulation. Cp and Pg will be purified at high level for in-vitro experiments. Cp fluorescence staining will be use to evidence infection for in-vitro experiments, and the morphology and maturation of Cp inclusions. The Core will culture macrophages from wild type and knockout mice, to be challenged with Cp and Pg for the study cytokine profiles and foam cell formation. DNA will be extracted from mice tissue (gingival, blood, lung, liver, spleen, and aorta), collected at different time points during infection, and microbial (Cp or Pg) DNA will be detected by PCR using primer sets specific for each microorganism. The number of organism (loads) contained in the tissue sample will be quantified using a new end point quantitative PCR method (a-PCR) that we have previously validated to study Ct infection and transmission. This assay has an excellent correlation with quantitative Ct culture. Cp and Pg loads will be quantified in in-vivo experiments. The presence of a threshold in the number of organisms in tissue that divide mice into responder vs. non-responders to bacterial antigen stimulation will be investigated. We hypothesize that the innate response is dependent on the organism load, and that un-infected mice and mice with low organism loads will have a lower response (or no response) compared with mice having higher organisms loads. We will study how organisms loads: 1) are modified by changes in innate immunity respond in infected gene-deficient mice (IL-p, IL-1R, ApoE, and LXR knockout mice); 2) influence the acceleration of atherosclerosis in mice infected with either Pg or Cp; and 3) influence oral bone loss in mice infected with Pg.